5-Fluorouracil (5-FU) is commonly used in the treatment of cancers, including cancers of the breast, head, neck, and digestive system. The efficacy of 5-FU as a cancer treatment varies significantly among patients. Clinically significant differences in systemic clearance and systemic exposure of 5-FU are often observed. [Grem, J. L. In Chabner, B. A. and J. M. Collins (eds.), Cancer Chemotherapy: Principles and Practice, pp. 180-224, Philadelphia, Pa., Lippincott, 1990)]. Furthermore, 5-FU treatment is severely toxic to some patients, and has even caused death. [Fleming et al. (1993) Eur. J. Cancer 29A: 740-744; Thyss et al. (1986) Cancer Chemother. Pharmacol. 16: 64-66; Santini et al. (1989) Br. J. Cancer 59: 287-290; Goldberg et al. (1988) Br. J. Cancer 57: 186-189; Trump et al. (1991) J. Clin. Oncol. 9: 2027-2035; Au et al. (1982) Cancer Res. 42: 2930-2937].
Patients in whom 5-FU is severely toxic typically have low levels of dihydropyrimidine dehydrogenase (DPD) activity [Tuchman et al. (1985) N. Engl. J. Med. 313: 245-249; Diasio et al. (1988) J. Clin. Invest. 81: 47-51; Fleming et al. (1991) Proc. Am. Assoc. Cancer Res. 32: 179; Harris et al. (1991) Cancer (Phila.) 68: 499-501; Houyau et al. (1993) J. Nat'l. Cancer Inst. 85: 1602-1603; Lyss et al. (1993) Cancer Invest. 11: 239-240]. Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the principal enzyme involved in the degradation of 5-FU, which acts by inhibiting thymidylate synthase [Heggie et al. (1987) Cancer Res. 47: 2203-2206; Chabner et al. (1989) In DeVita et al. (eds.), Cancer--Principles and Practice of Oncology, pp. 349--395, Philadelphia, Pa., Lippincott; Diasio et al. (1989) Clin. Pharmacokinet. 16: 215-237; Grem et al., supra.]. The level of DPD activity also affects the efficacy of 5-FU treatments, as 5-FU plasma levels are inversely correlated with the level of DPD activity [ligo et al. (1988) Biochem. Pharm. 37: 1609-1613; Goldberg et al., supra.; Harris et al., supra.; Fleming et al., supra.]. In turn, the efficacy of 5-FU treatment of cancer is correlated with plasma levels of 5-FU.
In addition to its 5-FU degrading activity, DPD is also the initial and rate limiting enzyme in the three-step pathway of uracil and thymine catabolism, leading to the formation of .beta.-alanine and .beta.-aminobutyric acid, respectively [Wasternack et al. (1980) Pharm. Ther. 8: 629-665] DPD deficiency is associated with inherited disorders of pyrimidine metabolism, clinically termed thymine-uraciluria [Bakkeren et al. (1984) Clin. Chim. Acta. 140: 247-256]. Clinical symptoms of DPD deficiency include a nonspecific cerebral dysfunction, and DPD deficiency is associated with psychomotor retardation, convulsions, and epileptic conditions [Berger et al. (1984) Clin. Chim. Acta 141: 227-234; Wadman et al. (1985) Adv. Exp. Med. Biol. 165A: 109-114; Wilcken et al. (1985) J. Inherit. Metab. Dis. 8 (Suppl. 2): 115-116; van Gennip et al. (1989) Adv. Exp. Med. Biol. 253A: 111-118; Brockstedt et a. (1990) J. Inherit. Metab. Dis. 12: 121-124; Duran et al. (1991) J. Inherit. Metab. Dis. 14: 367-370]. Biochemically, patients having DPD deficiency have an almost complete absence of DPD activity in fibroblasts [Bakkeren et al., supra.] and in lymphocytes [Berger et al., supra.; Piper et al. (1980) Biochim. Biophys. Acta 633: 400-409]. These patients typically have a large accumulation of uracil and thymine in their cerebrospinal fluid [Bakkeren et al., supra.] and urine [Berger et al., supra.; Bakkeren et al., supra.; Brockstedt et al., supra.; Fleming et al. (1992) Cancer Res. 52: 2899-2902].
Familial studies suggest that DPD deficiency follows an autosomal recessive pattern of inheritance [Diasio et al., (1988) supra.]. Up to three percent of the general human population are estimated to be putative heterozygotes for DPD deficiency, as determined by enzymatic activity in lymphocytes [Milano and Eteinne (1994) Pharmacogenetics (in press)]. This suggests that the frequency of homozygotes for DPD deficiency may be as high as one person per thousand.
DPD has been purified from liver tissue of rats [Shiotani and Weber (1981) J. Biol. Chem. 256: 219-224; Fujimoto et al. (1991); J. Nutr. Sci. Vitaminol. 37: 89-98], pig [Podschun et al. (1989) Eur. J. Biochem. 185: 219-224], cattle [Porter et al. (1991) J. Biol. Chem. 266: 19988-19994], and human [Lu et al. (1992) J. Biol. Chem. 267: 1702-1709]. The pig enzyme contains flavins and iron-sulfur prosthetic groups and exists as a homodimer with a monomer Mr of about 107,000 [Podschun et al., supra.]. Since the enzyme exhibits a nonclassical two-site ping-pong mechanism, it appears to have distinct binding sites for NADPH/NADP and uracil/5,6-dihydrouracil [Podschun et al. (1990) J. Biol. Chem. 265: 12966-12972]. An acid-base catalytic mechanism has been proposed for DPD [Podschun et al. (1993) J. Biol. Chem. 268: 3407-3413].
Because an undetected DPD deficiency poses a significant danger to a cancer patient who is being treated with 5-FU, a great need exists for a simple and accurate test for DPD deficiency. Such a test will also facilitate diagnosis of disorders that are associated with DPD deficiency, such as uraciluria. The present invention provides such a test, thus fulfilling these and other needs.